Plant Breeding 117, 485—487 (1998) © 1998 Blackwell Wissensehafts-Verlag, Berlin ISSN 0179-9541

Short Communication

Production of hapioids in , durum wheat and hexaploid crossed with pearl miUet

M. N. iNAGAKi^'^ and C. T. 'International and Wheat Improvement Center (CIMMYT), Lisboa 27, Colonia Juarez, Apartado Postal 6-641, 06600, Mexico, DF, Mexico; ^International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, 502324, Andhra Pradesh, ; ^Present address: Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki, 305-8686 Japan With 1 figure and 1 table Received February 1, 19981 Accepted March 4, 1998 Communicated by P. Ruckenbauer

Abstract Bennett 1994, Dusautoir et al. 1995, Inagaki and Tahir 1995, Pearl is an efficient alternative to maize as a pollen source for Inagaki et al. 1997b). Pearl millet {Pennisetum glaucum (L.) R. haploid production in bread wheat. To compare haploid production Br.) is an alternative pollen source for both efficient haploid frequencies in other species, the crossabilities of two genotypes production in bread wheat (Inagaki and Bohorova 1995, each of bread wheat, durum wheat and hexaploid triticale with four Inagaki and Mujeeb-Kazi 1995) and stable long-term pollen pearl millet genotypes and a maize control were examined. Embryos storage (Hanna and To will 1995, Inagaki and Mujeeb-Kazi were obtained from crosses of all three species with both pearl millet 1997). To compare haploid production efficiencies across a and maize. However, significant differences in crossability were found wider range of Triticeae, the crossabilities of bread wheat, among the three species (10.5-79.8% development and 1.4-15.8% embryo formation), as well as among genotypes of durum wheat (7.2- durum wheat and hexaploid triticale with pearl millet and maize 23.7% and 2.1-6.4%) and hexaploid triticale (0.3-20.6% and 0.1- were examined. 2.7%). Crossability of bread wheat with pearl millet was relatively high. Haploid were regenerated from crosses of all three species with materials: Two genotypes each from bread wheat ('Norin 61' and pearl millet. As in the case of maize crosses, low crossabilities of durum 'Attila'), durum wheat ('AItar-84' and 'Sora/2*Plata-12') and hexaploid wheat and hexaploid triticale with pearl millet can be attributed to the triticale ('Anoas-3/Tatu-4' and 'Jilotecpec-96') were used as female par- absence of D-genome . ents. Fresh pollen from three very early-flowering pearl millet inbred parents ('LGD-1-lO-B', 'H-77/833-2-P5' and '843B') of pearl millet Key words: Triticum aestivum — T. turgidum var. durum — mapping populations (Hash and Witcombe 1994) was used. In addition, Pennisetum glaucum — x Triticosecale — haploid — wide pearl millet pollen of'NEC-7006' stored for 14 months in liquid nitrogen crosses and fresh pollen of the single-cross maize 'CML-246 x CML- 242' were chosen as pollen sources (Inagaki et al. 1997a). Female parents were grown in the field. Temperatures at ear emergence were 28/8°C In recent years, the development of haploid production tech- (maximum/minimum). Pollen parents were grown in a greenhouse at niques for bread wheat {Triticum aestivum L., 2« = 6x = 42, 33/15°C (maximum/minimum temperatures). AABBDD), using elimination following wide crosses, has advanced rapidly (see Inagaki 1997 for a review). Crossing with pearl millet and maize: Spikes on detached tillers of bread wheat, durum wheat and hexaploid triticale were crossed with pearl One of the most successful techniques at present uses crosses millet and maize, and then cultured in a nutrient solution containing with maize {Zea mays L.) (Laurie and Bennett 1986, 1988) 40 g/1 sucrose, 100 mg/1 2,4-D and 8 ml/1 sulphurous acid. Five spikes followed by application of 2,4-dichlorophenoxyacetic acid (2,4- were used for each crossing treatment, with two replications. Statistical D) (Suenaga and Nakajima 1989). This technique can be data analyses on seed development and embryo formation percentages applied to diverse genotypes of bread wheat for stable haploid were performed after angular transformation. production (Inagaki and Tahir 1990) and used for instant pro- At 14 days after polhnation with pearl millet '843B' and maize 'CML- duction of recombinant inbred lines for both breeding purposes 246 X CML-242', immature embryos were aseptically excised from and genetic analyses. Wheat haploid production efficiency has developing on crossed spikes of cultured detached tillers, and also been improved significantly by pollen storage, detached- transferred on to Murashige and Skoog (1962) culture medium sup- tiller culture and hot-water emasculation, considerably reduc- plemented with 20 g/1 sucrose and 6 g/1 agarose. Procedures of detached- tiller culture and embryo rescue were as described by Inagaki et al. ing the labour and space required for crossing (Inagaki et al. (1997a, b). Regenerated plants were cytologically examined in squashed 1997a). root-tip preparations followed by modified Giemsa-staining procedures However, recent reports indicate distinct genotypic variation (Jahan et al. 1990). C-banded chromosomes were identified based on in the haploid production frequencies of durum wheat {Triticum the karyotypes of wheat and triticale (Friebe and Gill 1994, Lukaszew- turgidum L. var. durum, 2n = 4x = 28, AABB) and hexaploid ski and Gustafson 1983). triticale (x Triticosecale Wittmack, 2n = 6JC = 42, AABBRR) crossed with maize (Amrani et al. 1993, O'Donoughue and Seeds developed after pollination of pearl millet and maize

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f 7R Fig. 1: Seeds containing embryos (a, b and c) and somatic chromosomes of regenerated plants (d, e and f). which were obtained from crosses of bread wheat 'Attila'. durum wheat 'Sora/2*Plata-12' and hexaploid triticale 'Anoas-3/Tatu-4' with pearl millet "8433', respectively. Bars indicate 10//m

were filled with an aqueous solution and lacked solid among pollen sources, except for a pearl millet genotype ('LGD- (Fig. 1). Some immature embryos were found in developed 1-10-B') which produced little pollen. Embryos obtained in seeds. Mean seed development percentages (number of seeds pearl millet crosses were smaller than those of maize crosses developed/number of florets pollinated x 100) were 79.8, 15.5 and were often polyembryos developing multishoots (data not and 10.5% in bread wheat, durum wheat and hexaploid trit- collected). Significant differences in embryo formation per- icale, respectively. Mean embryo formation percentages (num- centage were found between the three Triticeae species and ber ofembryos formed/number of florets pollinated x 100) were among genotypes of each species. Mean percentages of plant 15.8,4.3 and 1.4% in bread wheat., durum wheat and hexaploid regeneration from embryos of these three species were 50.2% triticale, respectively (Table 1). There was no significant differ- and 47.9% in crosses with pearl millet '843B' and maize 'CML- ence in seed development and embryo formation percentages 246 X CML-242\ respectively.

Table 1: Seed development percentages (embryo formation in parenthesis) in bread wheat, durum wheat and hexaploid triticale crossed with pearl millet and maize

Pearl millet Maize 'LGD-1-lO-B' 'H-77/833-2-P5' •843B' 'NEC-7006' 'CML-246 X CML-242' Mean Bread wheat 'Norin 61' 73.8(29.4) 82.4(9.0) 77.6(18.0) 73.2(29.6) 72.5(17.0) 75.9a (20.6a) 'Attila' 72.8(0.3) 83.9(0.6) 94.5(28.2) 85.6(10.9) 81.0(14.9) 83.6a (11.0b) Durum wheat 'Sora/2*Plata-12' 3.0(0.7) 48.9(14.1) 16.7(8.5) 20.0(4.5) 29.7(4.3) 23.7b (6.4bc) 'Altar-84' 0.0(0.0) 25.6(8.1) 2.7(0.4) 7.6(2.0) 0.3(0.0) 7.2c (2. Ide) Hexaploid triticale 'Anoas-3/Tatu-4' 18.4(0.0) 41.0(1.4) 10.3(5.5) 18.0(5.2) 15.5(1.2) 20.6b (2.7cd) 'Jilotecpec-96' 0.0(0.0) 1.4(0.4) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.3c (O.le) Mean 28.0b(5.1b) 47.2a (5.6ab) 33.6ab(10.1a) 34.1a (8.7a) 33.2ab(6.2ab)

Means followed by the same letter within each trait in the column or row are not significantly different by the least significant difference (P = 0.05). Production of haploids in bread wheat, durum wheat and hexaploid triticale crossed with pearl millet 487

Cytological examination of regenerated plants (at least 10 Genotype effect and histological events in relation to embryo for- plants each from bread wheat and durum wheat, two plants mation after intergeneric crosses between durum wheat {Triticum from hexaploid triticale) chosen at random from crosses with turgidum Desf. var durum) and maize {Zea mays L.) or teosinte {Zea pearl millet '843B' showed that these plants were haploids (poly- mays L. ssp mexicana). J. Genet. Breed. 49, 353—358. haploids), carrying 21 chromosomes of bread wheat and hex- Friebe, B., and B. S. Gill, 1994: C-band polymorphism and structural rearrangements detected in {Triticum aestivum). aploid triticale and 14 chromosomes of durum wheat. Haploid Euphytica 78, 1—5. plants of bread wheat 'Attila' carried a lB/lR translocated Hanna, W. W., and L. E. Towill, 1995: Long-term pollen storage. Plant chromosome. A complement of chromosomes of both the A Breed. Rev. 13, 179—207. and B genomes were identified in haploid plants from durum Hash, C. T., and J. R. Witcombe, 1994: Pearl millet mapping popu- wheat 'Sora/2*Plata-12'. Seven chromosomes were found lations at ICRISAT. In: Witcombe, J. R., and R. R. Duncan (eds.). in those of hexaploid triticale 'Anoas-3/Tatu-4' (Fig. 1). Use of Molecular Markers in and Pearl Millet Breeding for In this study, stored pearl millet pollen was as effective as Developing Countries, 69—75. Overseas Development Admin- fresh pollen for haploid production in bread wheat, durum istration, London. wheat and hexaploid triticale. The crossability of each of these Inagaki, M. N., 1997: Technical advances in wheat haploid production three Triticeae species with pearl millet was similar to that with using ultra-wide crosses. JIRCAS J. (Japan) 4, 51—62. , and N. Bohorova, 1995: Factors affecting the frequencies of maize. Haploid plants were obtained in all three species crossed embryo formation and haploid plant regeneration in crosses of hex- with pearl millet. However, crossability with pearl millet aploid wheat with pearl millet. Breed. Sci. 45, 21—24. differed significantly among the three species and among geno- , and A. Mujeeb-Kazi, 1995: Comparison of polyhaploid pro- types of each species. Two bread wheat genotypes with com- duction frequencies in crosses of hexaploid wheat with maize, pearl pletely different pedigrees showed relatively high percentages millet and sorghum. Breed. Sci. 45, 157—161. of seed development and embryo formation, as previously , and- , 1997: Production of polyhaploids of hexaploid wheat reported (Inagaki and Mujeeb-Kazi 1995). Crosses of durum using stored pearl millet pollen. In: H.-J. Braun, F. Altay, W. E. wheat and hexaploid triticale with either pearl millet or maize Kronstad, S. P. S. Beniwal, and A. McNab (eds). Wheat: Prospects resulted in low seed development percentages and genotypic for Global Improvement, 327—333. Kluwer Academic Publishers, Dordrecht. differences in embryo formation percentage, as with crosses of , and M. Tahir, 1990: Comparison of haploid production fre- durum wheat with maize (Inagaki et al. 1998). These cross- quencies in wheat varieties crossed with Hordeum bulbosum L. and abilities may be increased by the addition of silver nitrate to maize. Jpn. J. Breed. 40, 209—216. detached-tiller culture solution (O'Donoughue and Bennett , and , 1995: Comparison of crossabiHties of tetraploid wheat 1994, Inagaki et al. 1998). As with maize, the lower crossability with Hordeum bulbosum and maize. Res. Commun. 23, 339— of durum wheat and hexaploid triticale with pearl millet may 343. be genetically attributed to their lack of the D-genome chro- , T. Nagamine, and A. Mujeeb-Kazi, 1997a: Use of pollen storage mosomes. Some D-genome chromosomes substituted in genetic and detached-tiller culture in wheat polyhaploid production through backgrounds of durum wheat and hexaploid triticale enhanced wide crosses. Cereal Res. Commun. 25, 7—13. crossability with maize (Inagaki et al. 1997b, 1998). In this , W. H. Pfeiffer, M. Mergoum, and A. Mujeeb-Kazi, 1998: Vari- ation of the crossabihty of durum wheat with maize. Euphytica, in study, pearl millet offered no advantage over maize as a fresh press. pollen source for haploid production in durum wheat and hex- , , , , and A. J. Lukaszewski, 1997b: Effects of D- aploid triticale. However, the shorter life cycle, smaller high- genome chromosomes on crossabiUty of hexaploid triticale (x Tri- tillering plants and greater ease of pollen storage (data not ticosecale Wittmack) with maize. 116, 387—389. shown) of pearl millet compared with maize are advantages Jahan, Q., N. Ter-Kuile, N. Hashmi, M. Aslam, A. A. Vahidy, and A. worth exploiting for bread wheat haploid production. In con- Mujeeb-Kazi, 1990: The status of the lB/lR translocation chro- clusion, a technique is required to improve the low crossability mosome in some released wheat varieties and the 1989 candidate of durum wheat and hexaploid triticale with pearl millet and/or varieties of . Pak. J. Bot. 22, 1—10. maize before wider application of haploid techniques in breed- Laurie, D. A., and M. D. Bennett, 1986: Wheat x maize hybridization. Can. J. Genet. Cytol. 28, 313—316. ing programmes for these species can be recommended. . and , 1988: The production of haploid wheat plants from wheat X maize crosses. Theor. Appl. Genet. 76, 393—397. Acknowledgements Lukaszewski, A. J., and J. P. Gustafson, 1983: Translocations and This report is a contribution from collaborative research involving modifications of chromosomes in triticale x wheat hybrids. Theor. CIMMYT, ICRISAT and JIRCAS. The authors thank V. Rosas, CIM- Appl. Genet. 64, 239—248. MYT, for his assistance on chromosome banding techniques. The Murashige, T., and F. Skoog, 1962: A revised medium for rapid growth manuscript was approved as ICRISAT JA no. 2236. and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473— 497. O'Donoughue, L. S., and M. D. Bennett, 1994: Durum wheat haploid References production using maize wide-crossing. Theor. Appl. Genet. 89,559— Amrani, N., A. Sarrafi, and G. Albert, 1993: Genetic variability for 566. haploid production in crosses between tetraploid and hexaploid Suenaga, K., and K. Nakajima, 1989: Efficient production of haploid and maize. Plant Breeding 110, 123—128. wheat {Triticum aestivum) through crosses between Japanese wheat Dusautoir, J. C, M. Coumans, F. Kaan, and F. Boutouchent, 1995: and maize {Zea mays). Plant Cell Rep. 8, 263—266.